This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
TUBB3 / Tubulin beta-3 chain · Western blot design guide
Design a Western Blot for TUBB3
Real validated TUBB3 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-TUBB3 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
TUBB3 Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~50.4 kDa | |
| Gel | 5–20% (catalog H01857) | |
| Positive control | Soft tissue (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated | |
| Caveat | Phosphorylation-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 2 isoform(s) |
Section 1
Real Curated TUBB3 Western Blot Protocols
The H01857 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | (1) Jurkat cell lysate, (2) Human kidney lysate, (3) 3T3 cell lysate, (4) Mouse brain lysate, (5) C6 cell lysate, (6) Rat heart lysate (H01857). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated (catalog H01857) |
| Gel % | 5–20% (catalog H01857) |
| Load | 50ug; reducing conditions (catalog H01857) |
| Transfer | a Nitrocellulose membrane at 150mA for 50-90 minutes (catalog H01857) |
| Membrane | Nitrocellulose membrane (catalog H01857) |
| Blocking | 5% Non-fat Milk/ TBS for 1.5 hour at RT (catalog H01857) |
| Primary antibody | H01857; use the WB datasheet starting dilution (standard starting point) |
| Primary incubation | overnight (catalog H01857) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 (catalog H01857) |
| Secondary incubation | 1.5 hour at RT (catalog H01857) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog H01857) |
| Detection | ECL (catalog H01857) |
Section 2
What Is the Expected TUBB3 Western Blot Band Size?
TUBB3 is predicted at 50.4 kDa; isoforms and modifications could affect migration, but no empirical band or feature-specific shift is established.
| Band near 50.4 kDa | Consistent with the UniProt predicted TUBB3 mass; confirm identity with an appropriate control |
| Single band near 50.4 kDa | Isoforms 1 and 2 may overlap; their migration is not established |
| Multiple bands near 50.4 kDa | Could include isoforms 1 and 2; separate migration is not established |
| Slightly shifted band | TUBB3 has phosphorylation and polyglutamylation sites, but their effect on migration is unestablished |
| Little or no band in a soluble fraction | Cytoskeletal localization may place TUBB3 in another fraction |
💡Expected TUBB3 appearanceThe UniProt predicted mass is 50.4 kDa; no empirical band size is supplied, so confirm a band near that size with band-identity controls.
| UniProt predicted mass | Provides a 50.4 kDa reference for TUBB3 |
| Full-length 450-residue chain | Provides the sequence underlying the predicted 50.4 kDa mass |
| Splice isoform 1 | May differ in size from isoform 2; direction and magnitude are unknown |
| Splice isoform 2 | May differ in size from isoform 1; direction and magnitude are unknown |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Cytoskeletal TUBB3 may be poorly recovered in the sampled fraction | Check the cytoskeletal fraction and use a positive-control lysate |
| Band higher than expected | An apparent shift could reflect TUBB3 modification, but its effect is unestablished | Compare with a TUBB3-positive control and verify antibody specificity |
| Band lower than expected | Isoform size differences are possible but unquantified | Compare with a positive control and check for degradation |
| Multiple bands | Isoforms 1 and 2 are listed, but distinct bands are unverified | Use a TUBB3-specific knockdown or other identity control |
| Weak or no signal | TUBB3 recovery may vary with cytoskeletal extraction | Check extraction, loading, and a positive-control lysate |
Experiment evidence
Western blot images showing TUBB3 band
H01857 · TUBB3
M01857-1 · TUBB3
M01857 · TUBB3
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for TUBB3 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for TUBB3 in Western blot, you can use soft tissue lysate, which HPA rates as highly positive.
Positive control: Soft tissue (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: A tissue lysate positive control and an HPA not-detected tissue negative control are feasible for this cytoskeletal protein.
HPA tissue expression evidence for TUBB3
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Soft tissue | peripheral nerve | High | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Cerebral cortex | neuronal cells | Medium | Protein (IHC) | HPA → |
| Colon | peripheral nerve/ganglion | Medium | Protein (IHC) | HPA → |
| Hippocampus | neuronal cells | Medium | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TUBB3 Western Blot Tips
Deeper troubleshooting and optimisation questions for TUBB3, answered from its protein features.
How should TUBB3 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could TUBB3 isoforms produce different bands?
Isoforms · UniProt lists isoforms 1 and 2; isoform 2 lacks residues 1–72 of the canonical sequence. An antibody against that region may miss isoform 2. Check which isoform its epitope covers before assigning bands; the feature does not establish where either isoform migrates.
Which TUBB3 phosphorylation sites matter when interpreting bands?
PTM · The supplied UniProt features list phosphoserine at canonical residue 172, attributed to CDK1, and at canonical residue 444. Keep these UniProt coordinates distinct from any antibody or paper numbering. Their presence does not establish a detectable mobility shift.
Does this guide establish induction of TUBB3?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
What transfer method to use for TUBB3 Western blot?
Transfer · Use the predicted 50.4 kDa mass to check transfer in the region of interest. The supplied features do not specify a membrane, transfer method, or settings. Verify transfer with a protein stain and adjust conditions if the region around 50 kDa transfers poorly.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the H01857 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should TUBB3 bands be quantified?
Quantitation · Define which isoforms the antibody detects before quantifying: isoform 2 lacks canonical residues 1–72. Quantify bands consistently across samples, and report separately any additional band whose identity has not been established. The supplied features do not identify an observed band position.
Should TUBB3 run exactly at its predicted mass?
Interpretation · The predicted mass is 50.4 kDa, but no observed band position is supplied. Use 50.4 kDa as a reference, not an exact expected migration position. The listed modifications alone do not establish a visible shift or explain any difference in apparent mass.
UniProt lists 5-glutamyl polyglutamate at canonical residue 438. Consider this site when evaluating an unexpected band or a modification-sensitive antibody, but do not assign a band to glutamylation from position alone. Confirm the antibody's epitope and modification specificity.
Check the antibody epitope against isoform 2's missing canonical residues 1–72. Compare candidate bands with the 50.4 kDa predicted mass, while treating the listed phosphorylation and glutamylation sites as possibilities requiring validation. No observed band position is supplied, and those features alone cannot identify an unexpected band.
UniProt lists 5-glutamyl polyglutamate at canonical residue 438. Consider this site when evaluating an unexpected band or a modification-sensitive antibody, but do not assign a band to glutamylation from position alone. Confirm the antibody's epitope and modification specificity.
Check the antibody epitope against isoform 2's missing canonical residues 1–72. Compare candidate bands with the 50.4 kDa predicted mass, while treating the listed phosphorylation and glutamylation sites as possibilities requiring validation. No observed band position is supplied, and those features alone cannot identify an unexpected band.
Boster reagents
TUBB3 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-beta Tubulin Rabbit Monoclonal Antibody, HRP Conjugated
Cat # H01857
Real WB data
Anti-beta III Tubulin TUBB3 Rabbit Monoclonal Antibody
Cat # M01857-1
Real WB data
Anti-beta III Tubulin TUBB3 Rabbit Monoclonal Antibody
Cat # M01857
All three listed rabbit monoclonal antibodies have TUBB3 Western blot images and report human, mouse, and rat reactivity. Their captions document specific tested lysates and bands; no publication evidence or independent validation is supplied.
Which to pick: Choose by the closest documented sample: M01857-1 shows human SH-SY5Y and SIHA cells plus rat and mouse brain, with a band near 50 kDa; M01857 shows HeLa; H01857 shows Jurkat, human kidney, 3T3, mouse brain, C6, and rat heart.
Source: BosterBio TUBB3 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q13509.
- Human Protein Atlas. TUBB3 tissue expression.
- PMC12932840 — target-verified WB comparison
- PMC5641069 — target-verified WB comparison
- PMC10531752 — target-verified WB comparison